Primary health services in Cape Town, South Africa.® MTB/RIF-based algorithm.To compare tuberculosis (TB) diagnostic yield in an existing smear/culture-based and a newly introduced Xpert® based algorithm. We identified the full sequence of sputum tests recorded in the electronic laboratory database for presumptive TB cases from 60 primary health sites during seven one-month time-points, six months apart. Differences in TB yield and temporal trends were estimated using a binomial regression model.TB diagnostic yield (the proportion of presumptive TB cases with a laboratory diagnosis of TB) was assessed using a non-randomised stepped-wedge design as sites transitioned to the Xpert® based algorithm. There was a decline in TB yield over time with a mean risk difference of -0.9% (95% CI -1.2% to -0.6%) (p<0.001) per time-point. When estimates were adjusted for the temporal trend, TB yield was 19.1% (95% CI 17.6% to 20.5%) in the smear/culture-based algorithm compared to 19.3% (95% CI 17.7% to 20.9%) in the Xpert® based algorithm with a risk difference of 0.3% (95% CI -1.8% to 2.3%) (p = 0.796). Culture tests were undertaken for 35.5% of smear-negative compared to 17.9% of Xpert® negative low MDR-TB risk cases and for 82.6% of smear-negative compared to 40.5% of Xpert® negative high MDR-TB risk cases in respective algorithms.TB yield was 20.9% (95% CI 19.9% to 22.0%) in the smear/culture-based algorithm compared to 17.9% (95%CI 16.4% to 19.5%) in the Xpert® based algorithm did not produce the expected increase in TB diagnostic yield. Studies are required to assess whether improving adherence to the Xpert® negative algorithm for HIV-infected individuals will increase yield. In light of the high cost of Xpert®, a review of its role as a screening test for all presumptive TB cases may be warranted.Introduction of an Xpert The World Health Organisation identified five priorities to accelerate progress towards achieving the 2015 United Nation’s Millennium Development Goals for tuberculosis (TB) [® MTB/RIF  (Xpert) as a replacement for smear microscopy for all presumptive TB cases in 2011. Xpert is recommended as the initial diagnostic test in individuals suspected of multidrug-resistant (MDR)-TB or human immunodeficiency virus (HIV)-associated TB [South Africa, which ranks second on the list of 12 countries contributing 75% of missed cases [iated TB .Mycobacterium tuberculosis (MTB) [Xpert has higher sensitivity than smear microscopy. A Cochrane Review of fifteen studies with Xpert replacing smear microscopy as the initial test, yielded a pooled sensitivity of 88% (95%CrI: 83% to 92%) and specificity of 98% (95% CrI: 97% to 99%) for detecting is (MTB) . In compis (MTB) . DespiteSputum culture, considered the gold standard in TB diagnosis and important in the diagnosis of smear-negative TB, is able to diagnose paucibacillary disease at concentrations as low as 10 bacteria per ml  comparedWhilst rapid, more sensitive molecular diagnostic tests such as Xpert have the technical capacity to address the limitations of smear and culture, very little has been reported on their use in routine operational conditions, particularly within diagnostic algorithms. Increased test sensitivity may not on its own translate into an increase in the proportion of bacteriologically confirmed TB cases. The role of the test within the algorithm, how effectively it is used, the use of follow-on tests such as culture and the number of presumptive TB cases screened may all play a role.The aim of this study was to compare the proportion of presumptive TB cases with a laboratory diagnosis of TB (TB diagnostic yield) in an existing smear/culture-based algorithm and a newly introduced Xpert-based algorithm within a routine operational setting. Other factors influencing the proportion of TB cases identified, including adherence to diagnostic algorithms and changes in the proportion of the population tested, were assessed.Policy Relevant Outcomes from Validating Evidence on ImpacT) evaluation (http://www.treattb.org/) to assess the impact of new molecular diagnostics on the diagnosis and treatment of tuberculosis.This study was part of a PROVE IT , 47% were co-infected. .™ MGIT™ 960) and drug susceptibility testing (DST) using the GenoType® MTBDRplus  line probe assay (LPA). Smear-negative, HIV-infected, low MDR-TB risk cases were required to submit a third specimen for culture.Provincial and municipal health authorities provided free TB diagnostic services at 142 primary health care (PHC) facilities in eight sub-districts. Prior to August 2011, a smear/culture-based algorithm was used . Policy From August 2011 to February 2013 an Xpert-based algorithm was phased into the eight health sub-districts with Xpert replacing smear microscopy . All preSamples were initially tested at a central national health laboratory. Three sub-district laboratories were established in 2013 to perform Xpert tests and smear microscopy. Results for all samples were entered into a networked electronic laboratory database.presumptive TB case was defined as an individual with pre-treatment sputum samples submitted for diagnostic purposes and a TB case as an individual with one or more smears positive and / or culture positive for MTB and / or MTB detected on Xpert.A Low MDR-TB risk was defined as ≤four weeks previous TB treatment and high MDR-TB risk as >four weeks previous TB treatment, from congregate settings or with a known MDR-TB contact.PHC site consisted of municipal and provincial health facilities linked to their satellite and mobile facilities and to each other if within a single geographic location (to account for shared diagnostic services).A We used a non-randomised stepped-wedge study design to assess TB yield in five groups of PHC sites over seven one-month time-points (T1 to T7) as they changed from using the smear/culture-based algorithm to the Xpert-based algorithm .We included 60 PHC sites (five of the eight sub-districts in Cape Town) that had implementation of either the smear/culture or the Xpert-based algorithm in October to December 2011. The three sub-districts transitioning in this quarter were excluded to avoid overlap between algorithms within the time-point. Other time-points were selected to also avoid overlap.The study population included all presumptive TB cases with sputum test results recorded in the electronic laboratory database in Cape Town for the selected facilities and time periods.The National Health Laboratory Services provided TB test data from the electronic laboratory database for 2010–2013. Data included patient demographic information, MDR-TB risk category, test type  and result.Data for each time-point and the preceding and following months (i.e. a quarter’s data) were imported into MS-SQL. The full sequence of tests for individuals at T1 to T7 were derived by identifying all tests done in that month as well as in the preceding and following months using a combination of name, surname, clinic folder number and age or birth date.TB diagnostic yield was calculated by dividing the number of TB cases identified (based on the full sequence of tests performed) by the total number of presumptive TB cases screened by algorithm and time-point.Adherence to the smear/culture-based algorithm was assessed by calculating the proportion of cases with two smear tests and the proportion of smear-negative cases with culture tests. Adherence to the Xpert-based algorithm was assessed by calculating the proportion of cases with an Xpert test and the proportion of Xpert-negative cases with culture tests.The proportion of presumptive TB cases tested in the five sub-districts was calculated by dividing the number of individuals tested in the quarter by the mid-year population estimates from Statistics South Africa .Descriptive statistics by algorithm are presented using frequencies, means and standard deviation. We used a binomial regression model, adjusted for site-level clustering, to estimate the differences in TB yield between algorithms and assess temporal trends. This model was used to assess the effect of MDR-TB risk on TB yield and to evaluate trends in testing over time. All analyses were undertaken using STATA 12 (StataCorp).The Health Research Ethics Committee at Stellenbosch University (IRB0005239) (N10/09/308) and Ethics Advisory Group at The International Union Against Tuberculosis and Lung Disease (59/10) approved the study. A waiver of informed consent was granted for use of routine data. The City Health Directorate, Western Cape Health Department and National Health Laboratory Service granted permission to use routine health data. Data sharing is possible following approval by the National Health Laboratory Service.The smear/culture-based algorithm included 24,000 presumptive TB cases and the Xpert-based algorithm 30,393 . Age andTB yield was 20.9% (95% CI 19.9% to 22.0%) in the smear/culture-based algorithm compared to 17.9% (95%CI 16.4% to 19.5%) in the Xpert-based algorithm with a risk difference of -3.0% (95% CI -4.3% to -1.7%) (p<0.001) . Adjustilow MDR-TB risk cases, TB yield in the smear/culture-based algorithm was 20.3% (95% CI 19.0% to 21.5%) compared to 17.1% (95% CI 15.1% to 19.1%) in the Xpert-based algorithm with a risk difference of -3.2% (95% CI -4.9% to -1.4%) (p<0.001). Amongst high MDR-TB risk cases, TB yield was 24.4% (95% CI 23.2% to 25.6%) and 22.1% (95%CI 21.0% to 23.3%) respectively with a risk difference of -2.3% (95% CI -3.8% to -0.7%) (p = 0.004).Amongst There was a declining trend in TB yield in both the smear-culture and Xpert-based algorithms . OverallWhen estimates were adjusted for the temporal trend, there was no significant difference between the algorithms: the proportion of TB cases identified in the smear/culture based algorithm was 19.1% (95% CI 17.6% to 20.5%) compared to 19.3% (95% CI 17.7% to 20.9%) in the Xpert-based algorithm with a risk difference of 0.3% (95% CI -1.8% to 2.3%) (p = 0.796) .low MDR-TB risk presumptive TB cases, 83.9% in the smear/culture-based algorithm had two smear tests and 35.5% of smear-negative cases had a culture test  than in the Xpert-based algorithm (32.8%) . The binThe trend in the proportion of the population tested over time varied from a high of 0.95% at T1 to a low of 0.80% at T5 and up to 0.89% at T7 . There wSouth Africa introduced Xpert as a replacement for smear microscopy for all presumptive TB cases in 2011. Since Xpert  has a hiA study in the rural Western Cape also found comparable TB yields amongst cases tested centrally through smear microscopy and Xpert . AnotherDemonstration studies done on a moderate scale and at sites selected for good performance tend to over-estimate test efficacy  and may We found a decline in TB yield over time; the decline in the smear/culture-based algorithm was continued under the Xpert-based algorithm, with no change point observed. To assess whether adherence to algorithms may have contributed to this temporal trend, we assumed “diagnostic equipoise” i.e. that sensitivity was not lower with Xpert than with two smears. We assessed the proportion of low and high MDR-TB risk cases with either two smears or an Xpert test at each time point. Both showed increases over time, indicating that although adherence to the Xpert-based algorithm was sub-optimal, cases were better-off than previously for initial testing.As HIV data for presumptive TB cases were not available for all time-points, we could not assess adherence to the second step of the algorithm . Overall, we found a higher proportion of smear-negative cases compared to Xpert-negative cases with culture tests. Since a higher proportion of HIV-infected cases may initially have been diagnosed by Xpert than by smear, the extent to which this may reflect poorer adherence to the algorithm cannot be ascertained. It is possible that poor adherence to the Xpert-negative algorithm for HIV-infected cases contributes to the temporal trend to some extent.The last few years has seen national efforts to intensify TB case-finding. As the proportion of the population tested increases, one would expect to find a decrease in TB yield. Our study found a minimal decline in the proportion of the population tested over time and this is therefore unlikely to have contributed to the temporal decline in TB yield.We do not have local TB prevalence estimates. A Cape Town study reported a decrease in TB prevalence following antiretroviral treatment roll-out in one community . It is pDiagnostic tests are rarely used in isolation. A unique aspect of this study is that we assessed TB diagnostic yield from each algorithm and not from individual tests. This reflected the variability that occurs in routine clinical practice, including both poor adherence to algorithms as well as the appropriate pursuit of a TB laboratory diagnosis after initial negative tests. Many presumptive TB cases are likely to undergo a sequence of tests and although this has been assessed in modelling studies ,25, it iWe used a stepped-wedge analysis, deemed appropriate where practical, financial or logistic constraints prevent the intervention being simultaneously introduced to all facilities . An advaThe extent to which results can be generalised is limited by the urban setting and good health and laboratory infrastructure, including access to liquid culture. Additional evidence is required from other settings particularly where culture is not routinely available.The failure to find an increase in TB notification rates following Xpert implementation has been attributed to high rates of empirical treatment ,20,27. TSeveral studies have reported other benefits with Xpert, including early TB ,28 and MDespite South Africa making great strides in expanding diagnostic services, including access to rapid tests through nation-wide implementation of Xpert, the proportion of TB cases diagnosed through laboratory tests in Cape Town has not increased. The historic smear/culture-based algorithm was as effective in identifying TB cases as the newly introduced Xpert-based algorithm. Additional studies are required to assess whether improving adherence to algorithms, particularly for Xpert-negative, HIV-infected individuals, will increase TB yield. Ultimately, difficult questions may need to be asked and answered about the future use of Xpert in resource-constrained settings where effective smear and culture-based algorithms are in place.


Plasmodiophora brassicae is one of the most common pathogens worldwide, the causal agent of clubroot disease in Brassica crops, resistance mechanisms to it are still only poorly understood. To study the early defense response induced by P. brassicae infection, a global transcriptome profiling of the roots of two near-isogenic lines (NILs) of clubroot-resistant (CR BJN3-2) and clubroot-susceptible (BJN3-2) Chinese cabbage (Brassica rapa) was performed by RNA-seq. Among the 42,730 unique genes mapped to the reference genome of B. rapa, 1875, and 2103 genes were found to be up- and down-regulated between CR BJN3-2 and BJN3-2, respectively, at 0, 12, 72, and 96 h after inoculation (hai). Functional annotation showed that most of the differently expressed genes are involved in metabolism, transport, signal transduction, and defense. Of the genes assigned to plant-pathogen interactions, 151 showed different expression patterns between two NILs, including genes associated with pathogen-associated molecular patterns (PAMPs) and effectors recognition, calcium ion influx, hormone signaling, pathogenesis-related (PR) genes, transcription factors, and cell wall modification. In particular, the expression level of effector receptors (resistance proteins), PR genes involved in salicylic acid (SA) signaling pathway, were higher in clubroot-resistant NIL, while half of the PAMP receptors were suppressed in CR BJN3-2. This suggests that there was a more robust effector-triggered immunity (ETI) response in CR BJN3-2 and that SA signaling was important to clubroot resistance. The dataset generated by our transcriptome profiling may prove invaluable for further exploration of the different responses to P. brassicae between clubroot-resistant and clubroot-susceptible genotypes, and it will strongly contribute to a better understanding of the molecular mechanisms of resistance genes of B. rapa against P. brassicae infection.Although  Plasmodiophora brassicae Wor. is a soil-borne, obligate, and biotrophic pathogen that attacks Brassica crops, leading to clubroot, and subsequent reductions in crop yield  in B. rapa -nucleotide-binding (NB)-leucine-rich repeat  genes in plants and triggers activation of the second mode of defense response, known as effector-triggered immunity (ETI). A number of defense responses are activated at the transcriptome level, including mitogen-activated protein kinase (MAPK) cascades, transcription factors, and hormone signaling  occurred 12 and 72 h, respectively, following inoculation with P. brassicae. The success of primary infection by P. brassicae was also observed in resistant genotypes of Brassica crops  at two stages of infection helps in understanding host–P. brassicae interactions.Brassica crops and P. brassicae, particularly with respect to mechanisms of resistance and the basal defense response. To date, several “-omics” approaches have been employed to examine the interactions between hosts and P. brassicae. Devos et al.  showed that, when compared with immune response in susceptible response, metabolic changes in the partial resistance response were reduced or delayed, and abnormal cell enlargement and proliferation were actively inhibited at 7 days post-inoculation on A. thaliana  synthesis and signal perception were involved in clubroot development. Chu et al.  and the clubroot-susceptible allele of crbcrb (BJN3-2) were inoculated with suspension of P. brassicae. CR BJN3-2 was developed by integration of the CRb gene from the CR Shinki DH line of Chinese cabbage into the Chinese cabbage inbred line BJN3-2 based on the marker-assisted selection  o et al. . The inoP. brassicae in the roots were observed using an inverted microscope  and imaged with digital single-lens reflex (DSLR) camera  every 2 h after inoculation until the secondary infection was established. The method of P. brassicae inoculation was the same as above. The roots were washed thoroughly with distilled water to remove the spores adsorbed on the surface, and then were transferred to a new 90 mm diameter glass petri dish with 30 ml distilled water for microscope observation. At each time point, five individual plants were examined, and three fields were observed for each sample.To determine the timing of primary and secondary infection of the two NILs, infection processes of P. brassicae inoculation. Roots of 30  plants were sampled and pooled for RNA extraction at each time point. The roots were washed in distilled water to remove the pathogen inoculum and immediately frozen in liquid nitrogen and stored at −80°C until use. To verify the success of infection, a pair of NILs was maintained in the culture room for 30 days after inoculation.Based on the observed infection stages, the primary and secondary infection were found to be happened at 12 and 72 h after inoculation (hai). Therefore, the roots of the CR BJN3-2 and BJN3-2 plants were sampled at 0, 12, 72, and 96 hai for analysis of the DEGs at the early stages of infection. Three independent biological replicates of both two NILs were performed for P. brassicae inoculation. Inoculation process and tissue sampling were exactly the same with the preparation of samples as above for RNA-seq. The pooled RNA was used to confirm the RNA-seq data results by quantitative RT-PCR (qRT-PCR).To cover the shortage caused by the lack of biological replication for RNA-seq, three independent biological replications were performed for RNA was extracted using TRIzol reagent  according to the manufacturer's instructions. RNA purity was determined using a Nanodrop spectrophotometer , 1% formaldehyde gel electrophoresis, and a 2100 Bioanalyzer . To eliminate the variability caused by human operation during the infection and sampling process, RNA from three biological replicates at each time point  were pooled.http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE74044 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE74044).cDNA library preparation and sequencing were conducted by the Biomarker Technology Company in Beijing, China. mRNA was isolated using beads with Oligo (dT), and then broken into short fragments after the addition of fragmentation buffer. These short fragments of mRNA were used as templates to synthesize first-strand cDNA with random primers. Second-strand cDNA was synthesized in a reaction containing DNA polymerase I, RNase H, dNTPs, and buffer. The resulting cDNA were then subjected to end-repair polymerase and mixed with Solexa adapters. Suitable fragments were recovered from an agarose gel. Next, PCR amplification was performed to enrich the purified cDNA template. Finally, all the eight libraries  were sequenced using an Illumina HiSeq™ 2000. The raw RNA-seq data has been deposited at GEO . Sequences were deemed to be significantly differentially expressed if the FDR was < 0.01 and there was at least a two-fold change  in RPKM between two libraries. Differential patterns of gene expression at the various time points are represented by Venn diagrams.Gene expression levels of all eight libraries were estimated by calculating read density as Reads Per Kilo base per Million mapped reads  using TopHat  and the rest of 19 DEGs were from the model of SA, JA, and ET signal pathway (Supplementary Table S1). Notably, 33 of the DEGs were from 0 hai.To validate the RNA-seq results, we performed qRT-PCR on 40 selected DEGs. Twenty-one of the selected DEGs were involved in resistant to P. brassicae. Each sample was divided into three independent biological and technical replicates. The comparative CT method of quantitation was used with B. rapa 18S rRNA and Actin as internal controls. The conditions for amplification were as follows: 15 min denaturation at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 25 s. Following amplification, melting curves were performed by increasing temperatures from 55 to 95°C at intervals of 0.5°C every 10 s to confirm the specificity of the PCR amplification.The qRT-PCR reactions were performed using SYBR Green SuperReal PreMix Plus (TIANGEN) on a Bio-Rad CFX 96 real-time PCR system . The primers were designed using Primer Premier 5.0 software and are listed in Supplementary Table S1. Analysis of gene expression was performed for all samples at 0, 12, 72, and 96 h after inoculation of CR BJN3-2 and BJN3-2 with P. brassicae between CR BJN3-2 and BJN3-2 were monitored after inoculation. There were no spores in the roots at 0 hai in both two lines, but spores were observed around the surface of the roots. Primary zoospores inside the root hairs were observed at 12 hai in both two lines. The presence of plasmodium in the cortical tissue was observed in BJN3-2 at 72 hai, indicating the beginning of secondary infection. Subsequently, numberous plasmodia were observed in the cortical tissue. In contrast, the secondary infection was not happened in CR BJN3-2 even after 72 and 96 hai. The same results were also obtained by Feng et al.  were all >80%, an indication that the accuracy and quality of the sequencing data were sufficient for further analysis. Approximately 78% of the sequenced reads (140 million mapped reads) were in alignment with the B. rapa genome reference sequence . An overview of the sequencing process is shown in Supplementary Table S2. The distribution of the expression level of the unigenes had similar patterns among the eight samples, suggesting there was no bias in the construction of the cDNA libraries . As shown in the Venn diagram in Figure All of the 3812 DEGs were annotated by comparing their sequences against five public databases, which permitted the assignment of several functional annotations (Supplementary Table S6): 3573 (93.73%) DEGs were found to have significant matches in the nr database, 2815 (73.85%) in the Swiss-Prot database, 1034 (27.12%) in the COG, 788 (20.67%) in the KEGG, and 3355 (88.01%) in the GO.A total of 3355 DEGs were assigned to three GO classes: biological process, cellular component, and molecular function. For the four time points, the cellular components most commonly represented were “cell” and “cell part,” whereas “catalytic activity” and “binding” were among the most commonly represented molecular function categories. The top five subcategories in the biological process class were “metabolic process,” “cellular process,” “response to stimulus,” “biological regulation,” and “developmental process” Figure . GO enriWe found 1034 DEGs that had a COG classification. Among the 25 COG categories, the largest group was “General function prediction only” followed by “Tanscription,” “Signal transduction mechanisms,” “Amino acid transport and metabolism,” and “Replication, recombination and repair.” However, we did not find DEGs in the categories of W (Extracellular structure) and Y , followed by “Ribosome” , “Plant–pathogen interaction” , “Oxidative phosphorylation” , and “Protein processing in endoplasmic reticulum” . These included 23 PRRs, 15 NBS-LRR containing R genes, 2 respiratory burst oxidase homologs (RBOH), 6 Ca2+ influx coding genes, 2 MAPK, 18 pathogenesis-related (PR) proteins, 20 hormone metabolism  related genes, 34 cell wall modification related genes, 10 chitinases, and 23 WRKY factors. To get a global view of DEGs involved in resistant to P. brassicae, the fold changes of these genes between the two NILs were represented via a heat map  genes, that were components of this defense signaling pathway  and nine genes associated with ET biosynthesis and signaling  were down-regulated in CR BJN 3-2 at different time points . For all 40 DEGs, including 33 identified at 0 hai, qRT-PCR detected the same expression tendency as the RNA-seq analyses  and discussed below.A total of 3812 DEGs were detected between CR BJN3-2 and BJN3-2. Among them, more genes were detected to be differentially expressed at 0 hai. Detection of more DEGs at 0 hai were also found in potato and soybean when they were challenged by phytophthora and bacterial leaf pustule, respectively,  were differentially regulated between CR BJN3-2 and BJN3-2 , flagellin sensing 2 (FLS2), chitin elicitor receptor kinase (CERK), and chitin elicitor-binding protein (CEBiP), which transduces signals that trigger PTI  and “RESISTANT TO P. SYRINGAE 5” (RPS5), respectively, were up-regulated at different time points in CR BJN3-2 (Supplementary Table S5). Previous work has shown that RPS4 confers specific resistance to Pseudomonas syringae pv. tomato carrying the avirulence gene AvrRPS4  were highly expressed at all time points in both NILs and no differences in the expression of these genes were found between CR BJN3-2 and BJN3-2 with the exception of MKK5, which was up-regulated at 72 hai in CR BJN3-2 , a R protein encoding the coiled-coil (CC)-NB-LRR domain, was up-regulated at 0, 12, and 72 hai in CR BJN3-2. Zhang et al. -, CML11-, CML24-, and CML38-like genes were all up- regulated at 0 hai in CR BJN3-2 compared to BJN3-2 , the putative functions of which were RBOH, exhibited up-regulation in CR BJN3-2  is a master regulator of the SA-mediated induction of defense genes . And several studies have reported that P. brassicae secretes a methyl transferase (PbBSMT) that reduces the accumulation of SA in infected roots and that the expression level of PbBSMT in spores and plasmodia was low, whereas transferase expression was high in clubroots of host plants  controlled by JA/ET-dependent pathways  were up-regulated. Chitin is main component of the cell wall in P. brassicae (Moxham and Buczacki, P. brassicae is PAMP, but seven chitin synthase of P. brassicae were found to be highly expressed during infection process (Schwelm et al., P. brassicae although it should be confirmed further.We identified 10 genes related to chitinase that were differentially expressed between the two NILs. Among them, 4 chitinase-related genes (P. brassicae in B. rapa.Five genes were down-regulated in the CR BJN3-2 genotype compared to the BJN3-2 genotype, including three PR3-type basic chitinase Figure . Down reBra004771 was up regulated at 72 hai, but down regulated at 96 hai in CR BJN3-2. There are also several reports showing that different types of chitinase operate against different pathogens in different plant species (Tabei et al., P. brassica and B. rapa need to be investigated further.The chitinase gene B. rapa during the early stages of P. brassicae infection. A total of 3812 differentially expressed genes were identified. The up-regulation of resistance protein-coding genes, SA-related genes and PR genes demonstrated that these components play a role in the interaction between B. rapa and P. brassicae. At the same time, our results also showed that the genes involved in the uncontrolled cell division and expansion that are induced by clubroot were suppressed to a greater extent in resistant NIL than in susceptible NIL. The present work revealed that many genes are differentially expressed between clubroot-resistant and clubroot-susceptible NILs when responding to P. brassicae infection, greatly expanded the vision of the genetic basis and improved our understanding of the molecular mechanisms underlying B. rapa resistance to P. brassicae.In this study, we first investigated the transcriptome response in the roots of clubroot-resistant and clubroot-susceptible NILs of JC analyzed the data, performed the experiments, and drafted the manuscript. WP participated in the data analysis and helped to draft the manuscript. BC helped analyzed the data. CZ contributed analysis tools and helped to draft the manuscript. ZP conceived the study, participated in its coordination, and helped to draft the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Following chemotherapy and/or the administration of growth factors, such as granulocyte-colony stimulated factor (G-CSF), hematopoietic stem cells (HSC) mobilize from bone marrow to peripheral blood. This review aims to systematically present the structure of the HSC “niche” and elucidate the mechanisms of their mobilization. However, this field is constantly evolving and new pathways and molecules have been shown to contribute to the mobilization process. Understanding the importance and the possible primary pathophysiologic role of each pathway is rather difficult, since they share various overlapping components. The primary initiating event for the mobilization of HSC is chemotherapy-induced endogenous G-CSF production or exogenous G-CSF administration. G-CSF induces proliferation and expansion of the myelomonocytic series, which leads to proteolytic enzyme activation. These enzymes result in disruption of various receptor-ligand bonds, which leads to the disanchorage of HSC from the bone marrow stroma. In everyday clinical practice, CXC chemokine receptor-4 (CXCR4) antagonists are now being used as mobilization agents in order to improve HSC collection. Furthermore, based on the proposed mechanisms of HSC mobilization, novel mobilizing agents have been developed and are currently evaluated in preclinical and clinical studies. The deep knowledge and understanding of HSC mobilization will give insight into the mechanisms of “poor mobilization” and moreover may help in developing new mobilizing agents.Autologous hematopoietic stem cell transplantation (ASCT) is a widely used therapeutic strategy in the treatment of multiple myeloma and relapsed/refractory lymphomas. It can offer long-term disease control or even cure in a substantial proportion of patients. The prerequisite of ASCT is a successful and adequate stem cell mobilization and collection. Initial observations regarding the steady state circulation of hematopoietic stem cells (HSC) in the blood led to the study of HSC kinetics after the administration of chemotherapy with or without growth factors. Thus, nowadays, collection of HSC from the bone marrow (BM) has been neglected at least in the autologous transplantation setting and has been largely replaced by peripheral blood stem cell collection through cell separators. Patients who fail to collect ≥2.0 × 10The term HSC was introduced for the first time by Alexander Maximov in 1909 , 3. The Anatomically, the niche is located in close proximity to the endosteum and is supported by a variety of cells and molecules referred as “stroma.” The main representatives of the stroma are bone tissue cells (osteoblasts (OB), osteoclasts (OC), osteomacrophages (OMAC), chondrocytes, fibroblasts, and fat cells), reticuloendothelial cells , endothelial cells, as well as mesenchymal stem cells (MSC), myocytes, and cells of the autonomous nervous system. Noncellular stromal elements include the extracellular matrix (ECM), collagen, and minerals .+CD45− osteoblastic cells” (SNO) [8. The third niche type refers to a microenvironment rich in oxygen, with low calcium content, consisting of the vascular sinusoidal endothelial cells. Among them, the BM-derived endothelial cells (BMEC) are in close proximity to Nes+ MSC and CAR [+, CD38−, CD45RA−, and CD90+. However the ultimate proof of their “stemness” comes from experimental in vivo assays, such as long-term repopulating (LTRA), competitive repopulation unit (CRU), SCID repopulating cell (SRC), and limiting dilution assays [Three types of niches have been recognized: the endosteal (osteoblastic), the reticular, and the vascular . The former is located at the endosteum and consists mainly of the “spindle-shaped N-cadherins” (SNO) , 7. The  and CAR . HSC repn assays .0 phase of the cell cycle, while only 10% of their progenies are quiescent. It has been shown that approximately 30% of the quiescent HSC divide every 145–193 days, while a more active subpopulation does so every 28–36 days [The stem cell niche is essential for the quiescence of HSC. More than 70% of them are in the G–36 days . These tβ, the ubiquitin/proteasome pathway, and signaling pathways through ATM, P13K-Akt, mTOR, Mdm2/p53, osteopontin, and Notch and Wnt [LT-HSC protect themselves from DNA damage by limiting the number of their cellular divisions. The main DNA-repair mechanism of HSC is the nonhomologous end joining, NHEJ . In addi and Wnt –21. In a and Wnt , 23.It is obvious that the description of the aforementioned three types of niches is undertaken for understanding purposes. They do not represent different anatomical compartments, since bone and vessels are in close proximity to each other . The “niA concise presentation of the main cells, receptors, and ligands that interact with each other during the process of mobilization is shown in In 1976 Richman et al. described an increase of HSC in the blood of patients who had undergone chemotherapy . Later, It directly activates OMAC and macrophages, a fact that downregulates neighboring SNO, Nes+ MSC, and CAR, leading to reduced production of stromal-derived factor-1 (SDF-1) by these latter cells , 30. RedThe hyperplastic myelomonocytic series (through G-CSF) secrete a large variety of proteases, which induce proteolytic cleavage/clearance of SDF-1, leading to the release of the CXCR4 receptors on HSC and their subsequent liberation from the BM stroma. The activity of the proteases is further assisted by the cleavage of protease inhibitors . The mosThe same proteolytic G-CSF-induced mechanism is responsible for the degradation of VCAM-1, fibronectin, and OPN, leading to reduced cellular adhesion of HSC through their receptor VLA-4 to BM stroma , 37.G-CSF evokes a shift of HSC to more central locations in BM, close to the vascular endothelium due to increased oxygenation needs of the HSC . HSC movG-CSF reacts as a potent noradrenalin reuptake inhibitor. An additive effect of G-CSF and tricyclic antidepressants in HSC mobilization has been shown . Vice veG-CSF promotes proliferation and maturation of the myeloid series, while at the same time it induces substantial changes in the bone marrow stroma, leading to the rise of HSC in the circulation by 60 times compared to baseline . Only ceThe two basic G-CSF-induced mechanisms of SDF-1/CXCR4 and VCAM-1/VLA4 disruption have a synergistic effect. Coadministration of G-CSF and CXCR4 inhibitors or G-CSF and anti-VLA4 antibodies results in additive and more potent HSC mobilization compared to G-CSF alone .ζ (PKC-ζ) [ζ induces motility, adhesion and survival of CD34+ cells and MMP-2, and MMP-9 secretion. In addition, CXCR4 expression is dependent on stem cell factor (SCF) [SDF-1 (CXCL12) is a CXC chemokine, secreted by various BM stromal cells, such as CAR, Nes+ MSC, osteoblasts, and endothelial cells. The interaction of SDF-1 with its receptor CXCR4 on HSC plays a key role in HSC retention and trafficking. The expression of CXCR-4 on HSC is enhanced through a signaling cascade involving cAMP, phosphatidylinositol-4,5-biphosphate 3 kinase (PI3K), a number of GTPases, and atypical protein kinase C isoform  (PKC-ζ) . PKCζ inor (SCF) . SDF-1 ior (SCF) . As alreor (SCF) .The recently introduced mobilizing agent CXCR4 inhibitor plerixafor (AMD3100) disrupts the SDF-1/CXCR4 axis in a synergistic way to G-CSF. The following hypotheses have been proposed for its mechanism of action . (a) The+ cells. The proportion of the most primitive HSC (CD34+ CD133+ CD38−) has been reported higher in grafts from patients with lymphomas mobilized with chemotherapy/G-CSF plus plerixafor [+ cells [+ cells may contain a wider range of lineage progenitors and hypothesize that CD34+ cells mobilized with G-CSF may result in faster neutrophil recovery, while those mobilized with plerixafor or plerixafor plus G-CSF may have more rapid B-cell and T-cell recovery [Comparing the differences between G-CSF- and plerixafor-induced mobilization, it should be noted that CXCR4 antagonists lead to a more rapid mobilization. Additionally, CXCR4 antagonists do not induce either myeloid hyperplasia or proteolysis. It has been shown from experiments in primates that myeloid hyperplasia is not necessary for mobilization . Recent erixafor . Fruehau[+ cells . Donahuerecovery . Prospecrecovery , 49.1 receptors leads to impaired mobilization after CXCR4 inhibitors in mice, while intravenous administration of S1P1 agonists induces the opposite effect [2 (PGE2) regulates the expression of CXCR4 on HSC and aids SDF-1 in attracting them to the BM [The SDF-1/CXCR4 axis seems to be influenced by sphingolipids, with the main representative being sphingosine-1-phosphate (S1P), which is abundantly produced by red blood cells, activated platelets, and endothelial cells . Currente effect , 53. Thee effect . Thus boe effect . On the o the BM .β1 integrin very late antigen-4 (VLA-4) is expressed by HSC and facilitates their adhesion on BM stroma through interaction with vascular cellular adhesion molecule-1 (VCAM-1), fibronectin, and OPN. Studies in mice, primates, and humans have shown that the administration of the anti-VLA-4 monoclonal antibody  or blockade of its ligands leads to a potent mobilization effect [The VLA-4/VCAM-1 axis: the n effect , 28, 58.+ cells, which in turn results in membranal CD44 cleavage and HSC egress from the BM [CD44 (HCAM): CD44 has many isoforms. Among these, CD44s, the smallest one, is the most common one expressed on HSC. The major ligand of CD44 is hyaluronic acid (HA), a component of ECM. The most HA-rich regions of the bone marrow stroma are the endosteum and the sinusoidal endothelium, the sites that are considered the niches of the most primitive HSC. CD44/HA interactions are essential for homing of HSC in the BM. Membranal CD44 cleavage on HSC is associated with mobilization and depends on the equilibrium of a membrane-bound proteolytic enzyme  and its inhibitor RECK. G-CSF administration leads to increased expression of MT-1 MMP on CD34m the BM , 60. HomAdhesion molecules are surface antigens that facilitate cell-cell and cell-extracellular matrix (ECM) interactions, through their respective receptors. They play a major role in inflammation, mediating trafficking, endothelial rolling, adhesion, and extravasation of leukocytes and lymphocytes. They are essential for cellular immune responses, normal hematopoiesis, and differentiation, as well as for the organization of cells within tissues during ontogenesis. They are widely distributed on hematopoietic and nonhematopoietic cells . AccordiStem cell factor (SCF) is an important chemokine produced by SNO and CAR. Its active form binds to c-kit on the surface of HSC. C-kit (CD117) is a type III tyrosine kinase receptor and is also expressed by endothelial progenitor cells and MSC. The SCF/c-kit axis plays an important role in embryonic hematopoiesis and cellular differentiation. SCF has been shown to augment engraftment in mice through increased CXCR-4 expression on HSC . ExperimIt is nowadays widely accepted that bone tissue plays an important role in maintaining and regulating the HSC niche. Bone remodeling is a complex process and its consequences on HSC retention and mobilization are an evolving field .κB ligand (RANKL) is a cytokine that belongs to the tumor necrosis factor superfamily. It is produced by OB, other stromal cells, and activated T-lymphocytes. It stimulates osteoclastic differentiation, maturation, and activation. RANKL upon binding to its receptor RANK (on OC) stimulates osteoclastic activity. RANKL may also bind to osteoprotegerin (OPG). OPG is also produced by OB and acts as a soluble decoy receptor of RANKL that prevents the latter from binding to RANK. Thus the regulation of osteoclastic activation is dependent on RANKL/OPG balance.Receptor activator of NF-The administration of RANKL in mice resulted in an expected increase of osteoclastic activity, but also a simultaneous HSC mobilization, was observed . During In an effort to elucidate the role of bone tissue in human HSC mobilization, we undertook a study of various biochemical markers of bone remodeling, osteoclast/osteoblast regulators, and angiogenic cytokines in the process of HSC mobilization. We studied 24 patients, 10 with multiple myeloma, 5 with Hodgkin's Lymphoma, and 9 with non-Hodgkin's lymphoma who were mobilized with G-CSF +/− chemotherapy . We founCD45, known as leukocyte common antigen, is expressed by all leukocytes, including HSC. It is a transmembrane protein tyrosine phosphatase. Through dephosphorylation of Src kinase, it is involved in signal transduction pathways, regulating several cell processes. CD45 has been mainly studied in lymphocyte maturation, proliferation, and activation. Recent data have demonstrated a pivotal role of CD45 in HSC motility and microenvironmental regulation in both mice and human. From CD45 knockout mice experiments and CD45 treated human HSC in xenotransplantation experiments, it has been shown that CD45 is essential for HSC mobilization. G-CSF leads to increased expression of CD45 in bone marrow mononuclear cells, which in turn correlates with HSC release from the bone marrow. HSC from mice lacking CD45 show reduced mobilization, impaired ability to cross extracellular matrix barriers, and a hyperadhesive phenotype. In addition CD45 is required for normal osteoclastic development and function. CD45 negative osteoclasts display abnormal morphology and have a reduced ability to form multinucleated cells. Moreover they secrete lower amounts of MMP-9 and have a reduced bone resorbing activity. CD45 negative osteoclasts display a reduced response to RANKL resulting in poor HSC mobilization. Thus CD45 is a paradigm of the concurrent regulation of hematopoiesis and the microenvironment , 69.α and HIF-1β. Under conditions of adequate oxygenation, HIF-1α is hydroxylated and is recognized by Von Hippel-Lindau protein, leading to its proteasome-mediated degradation [α is prevented, and HIF-1α is stabilized. The full heterodimeric HIF-1 binds to hypoxia response elements of the genome, resulting in the transcription of several genes, including VEGFA in the BM sinusoids [The main regulator of hypoxia in the endosteal niche is HIF-1, consisting of HIF-1radation . Under hinusoids . VEGF reα are necessary for mobilization [It has been shown that activation of m-TOR and the resultant increase of reactive oxygen species (ROS) in the BM lead to the egress of HSC, whereas the inhibition of mTOR with rapamycin has the opposite effect. It appears that a “critical” level of ROS and HIF-1lization .As already stated, the main initiating molecule of the HSC mobilization process is exogenous or chemotherapy-induced endogenous G-CSF. For many years, the administration of G-CSF with or without chemotherapy has been the mainstay of mobilization in every day clinical practice. Ifosfamide-containing (IGEV and ICE) or platinum-containing (ESHAP and DHAP) regimens with G-CSF are usually applied in lymphomas –75. Thes+ cells collected per kg of body weight. Adequate number is considered the dose of HSC that ensures rapid and sustained long-term hematopoiesis after the administration of myeloablative chemotherapy and HSC infusion. The optimal dose of HSC is 5 × 106/kg, with little clinical benefit with doses 5–8 × 106/kg and no further improvement with grafts containing >10 × 106/kg CD34+ cells [6/kg.The endpoint of every mobilization attempt is the collection of adequate numbers of HSC, reflected practically by the absolute number of CD344+ cells . The lowStill, there are a substantial proportion of patients who fail to mobilize and collect adequate numbers of HSC and consequently cannot proceed to autologous stem cell transplantation. The inability of adequate HSC mobilization is associated with HSC reserve exhaustion and disruption of BM stroma . HoweverRisk factors for poor mobilization include age >65 years, bone marrow cellularity <30%, bone marrow infiltration, multiple previous chemotherapy regimens, especially alkylating agents, fludarabine, >4 cycles of lenalidomide, and previous extended field radiotherapy including the pelvis , 81.6/kg CD34+ cells through three consecutive days of apheresis, after the administration of an adequate mobilization regimen (G-CSF monotherapy at a dose of ≥10 μg/kg/day or chemotherapy + G-CSF ≥ 5 μg/kg/day) [+ cells < 20/μL on the day of the expected peak are also considered poor mobilizers [μg/kg, peak levels of CD34+ cells are expected on the 5th-6th day after the initiation of G-CSF. After ifosfamide-containing regimens + G-CSF, the peak day is usually the 12th-13th day since chemotherapy initiation, whereas the corresponding day for platinum-containing chemotherapy ranges between the 15th–17th day.There is not a widely accepted definition of the “poor mobilizer.” For this purpose, the Italian group of apheresis has proposed criteria not only for the definition, but also for the early recognition of the poor mobilizer . Accordi/kg/day) . Howeverbilizers . The day+ cell collection (≥5 × 106/kg) in the G-CSF plus plerixafor arm compared to the G-CSF plus placebo arm (19.6%). In addition 86.7% of the patients mobilized with G-CSF plus plerixafor reached the minimum target of ≥2 × 106/kg CD34+ cells versus 47.3% of those who received G-CSF plus placebo. The number of apheresis sessions was also significantly lower in the combination arm. The median fold increase in circulating CD34+ cell count from day 4 to day 5 was 5.0 in the plerixafor treated patients and 1.4 in the placebo treated patients. Similarly in the myeloma study [6/kg CD34+ cells in ≤2 apheresis procedures versus 34.4% of the G-CSF plus placebo group. Plerixafor has an excellent safety and tolerability profile.Plerixafor is a reversible CXCR4 antagonist and its mechanisms of action have been already described. It has been approved in both the US and the EU for the mobilization of patients with lymphoma and myeloma. With its use in combination with G-CSF, approximately 70% of poor mobilizers may succeed in the collection of adequate numbers of HSC in order to proceed to ASCT . The supma study  71.6% ofμg/kg/day × 4 days and plerixafor is administered at a dose of 240 μg/kg on the fourth day, 9–11 hours before collection on the 5th day; however with this strategy, significant time is lost between the mobilization attempts, which might be crucial for some patients with aggressive disease; in addition, the repetition of the whole procedure is cumbersome for the personnel and inconvenient for the patient; (iii) the preemptive—“just in time”—use of plerixafor seems to be the most cost-effective strategy: the proper mobilization regimen according to the patient's disease status is chosen. If CD34 counts in peripheral blood on the day of the expected peak are suboptimal (<20/μL), plerixafor is administered prior to HSC collection. With this latter strategy >90% of the patients can achieve grafts with >2 × 106/kg CD34+ cells [+ cells can be achieved in 3–8 hours after its administration, which may be more convenient for organizing the procedure of apheresis [The high cost associated with plerixafor requires a stringent protocol with well-defined criteria for its proper use , 88, 91.4+ cells . Anotherpheresis .Although plerixafor rescues a significant proportion of patients from failing collection of HSC, there is still a 35% possibility of its failure. Towards this aim, new mobilizing agents are being tested either in preclinical or clinical studies.6/kg CD34+ collected cells for the highest dose used. Moreover the number of apheresis procedures was reduced with the highest doses. This agent warrants further evaluation, since it also shows antimyeloma activity [Agents targeting the SDF1/CXCR4 axis: POL6326  is a potent selective CXCR4 inhibitor, which has shown considerable efficacy as monotherapy in newly diagnosed multiple myeloma patients . Its excactivity , 96. Othactivity  and NOX-activity . The lat+ yield of 23.2 × 106/kg in a median of 1 apheresis session [Proteasome inhibitors: bortezomib is nowadays considered among the leading therapeutic drugs in multiple myeloma. Surprisingly, in animal studies, bortezomib proved to be a potent mobilizing agent, increasing significantly CFU-Cs compared to placebo. In addition, a synergistic effect of bortezomib with G-CSF, plerixafor, and chemotherapy was evident . The mos session .Parathormone (PTH): Brunner et al. showed that PTH induces a significant increase of progenitor cells in the peripheral blood (1.5- to 9.8-fold) of mice. The authors postulated that this activity relates to endogenous release of G-CSF . PTH wasVLA-4/VCAM-1 Inhibitors. The importance of VLA-4/VCAM-1 axis has been already extensively described. Papayannopoulou and Nakamoto were among the pioneers in studying the effect of the VLA-4/VCAM-1 axis disruption in HSC mobilization. They showed that the administration of anti-VLA-4 antibodies resulted in the mobilization of HSC into the circulation [α4 subunit of VLA-4, approved for treatment of multiple sclerosis (MS) and Crohn's disease. In patients with MS treated with Natalizumab, a gradual increase in circulating CD34+ cells has been observed with a peak being present in 3-4 days after treatment. However the persistence of CD34+ cells in the circulation for an extended time period may be problematic in clinical practice [culation . Moreoveculation . Althougpractice , 105. Thβ (CXCL2), a chemokine whose exact mechanism is unclear [Other novel agents currently evaluated in preclinical studies are FG-4497 that stabilizes HIF-1 through inhibition of its hydroxylation , Groβ (C unclear , and S1P unclear .The mechanisms of HSC mobilization are overlapping and not fully elucidated yet. Experimental studies have given light into many aspects of this cascade of events. However, we are still far away from establishing an integrated model of the mechanisms that control the equilibrium of HSC between quiescence and mobilization. Laborious research is mandatory for the development of newer agents that might render HSC mobilization and collection possible for all patients.


Roseovarius tolerans EL-164 is a member of the Roseobacter clade, a group of marine bacteria within the Alphaproteobacteria. It produces different N-acylhomoserine lactone (AHL) autoinducers as well as five AHL-related but functionally different compounds, the N-acylated alanine methyl esters. The size of the draft genome is 3,749,755 bp. Roseobacter clade (family Rhodobacteraceae) is a group of marine bacteria within the Alphaproteobacteria. Roseovarius is one of the largest genera in the clade, which comprise four different lineages  and was one of the first described species of the genus  autoinducers with side chain lengths of 14 and 16 carbon atoms, and with and without an unsaturation . NAMEs are novel compounds, which are related to AHL autoinducers but show no quorum-sensing or quorum-quenching activity (De novo assembly with SPAdes version 2.5.0 (The lineages . Roseovahe genus . R. tole present . R. toleactivity . Chromosactivity . PreparaLGVV00000000. The version described in this paper is the first version, LGVV01000000.This whole genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession


Human decision-making arises from both reflective and reflexive mechanisms, which underpin goal-directed and habitual behavioural control. Computationally, these two systems of behavioural control have been described by different learning algorithms, model-based and model-free learning, respectively. Here, we investigated the effect of diminished serotonin (5-hydroxytryptamine) neurotransmission using dietary tryptophan depletion (TD) in healthy volunteers on the performance of a two-stage decision-making task, which allows discrimination between model-free and model-based behavioural strategies. A novel version of the task was used, which not only examined choice balance for monetary reward but also for punishment (monetary loss). TD impaired goal-directed (model-based) behaviour in the reward condition, but promoted it under punishment. This effect on appetitive and aversive goal-directed behaviour is likely mediated by alteration of the average reward representation produced by TD, which is consistent with previous studies. Overall, the major implication of this study is that serotonin differentially affects goal-directed learning as a function of affective valence. These findings are relevant for a further understanding of psychiatric disorders associated with breakdown of goal-directed behavioural control such as obsessive-compulsive disorders or addictions. When choosing an action, we use multiple strategies to obtain potential reward and to avoid potential punishment. Studies on humans and other animals suggest the existence of ‘reflective' or goal-directed responses that depend on prospective consideration of future actions and their consequent outcomes in contrast to ‘reflexive' or habitual responses that relies on retrospective experience with good and bad outcomes.4 Specifically, a model-based strategy was linked to the goal-directed behavioural control, whereas a model-free strategy, which presumes choices based on previously reinforced actions, suggests shared similarities with habitual control.3 Nevertheless, it is likely that habitual behaviour exceeds a simple reinforcement learning model-free mechanism.5Computationally, two behavioural control systems have been proposed to arise from different learning algorithms, model-based and model-free learning.7 Chemical neuromodulation of these systems by the ascending monoaminergic projections has only recently been addressed. Namely, numerous studies have focused on the role of dopamine (DA) as a signal of positive prediction error in model-free learning.10 Interestingly, administration of the dopaminergic precursor, levodopa, to healthy volunteers shifted behavioural performance to a model-based over a model-free strategy.11These two (often competitive) behavioural control strategies may depend on distinct neuronal systems, and more specifically on limbic (model-free) and on cognitive (model-based) corticostriatal circuits.12In contrast, the question whether serotonin , another monoamine neurotransmitter, influences the degree to which behaviour is governed by either model-based or model-free systems has not been previously addressed. Serotonin is sometimes considered to be in an opponent, or alternatively in a synergistic, functional relationship with brain DA with respect to behavioural choice.14 Consequently, 5-HT might influence the degree to which behaviour is governed by either model-based or model-free systems in both reward and punishment conditions.Recent data show that manipulation of 5-HT can selectively produce effects on both appetitive and aversively motivated behaviour.16 In contrast, low serotonin increased delayed reward discounting.17 Consequently, lower serotonin neurotransmission may affect the prospective consideration of behavioural choices and consequently shift the balance between two behavioural controllers towards model-free behaviour. Under punishment, lowering of serotonin levels promoted lose-shift associative learning19 and reduced the pavlovian inhibitory bias to aversive stimuli,21 which potentially might shift the balance towards goal-directed behaviour.In particular, selective activation of 5-HT neurons of the raphé nucleus promoted long-term optimal behaviour by facilitating waiting for the delayed rewards.22 that dissociated the reward and punishment conditions. This task discriminates model-based and model-free behavioural strategies  mixture in a randomised, placebo-controlled, double-blind order . They we25 The obtained values were entered in repeated measures analysis of variance (ANOVA) with time as a dependent factor and group as an independent factor.TRP was depleted by ingestion of a liquid amino acid load that did not contain TRP but did include other large neutral amino acids (LNAAs)  to one of two pairs of stimuli in stage 2. Each of the four second-stage stimuli was associated with probabilistic £1 monetary reward (in the reward version of the task) or loss (in the punishment version of the task), with probability varying slowly and independently over time (0.25 to 0.75). The punishment version had a different colour code and stimuli set on the first and second task stages. Both versions of the task had the same transition probabilities and dynamic range of the reward or the punishment probability. Participants completed 201 trials for each task version divided into three sessions. The order of performance of the task versions was counterbalanced and the two versions were separated by at least 1 h.We used the two-stage decisional task with separate reward and punishment conditions . The rewBefore the experiment, all subjects underwent the self-paced computer-based instructions explaining the structure of the task and providing practice examples. Overall, the subjects were instructed to win as much money as they could in the reward version and to avoid monetary loss in the punishment version of the task. Participants were told that they would be paid for the experiment depending on their cumulative performance in both task versions. They were paid a flat amount of £60 at the end of the experiment.Before analysis, we applied the arcsin transformation to the non-normally distributed behavioural variables and log transformation to the reaction times that allowed the normalisation of the data, with Shapiro–Wilk test <0.05 for all variables in both groups.For both versions of the task, we performed two types of analyses: one a factorial analysis of shifting and staying behaviour , and the second the fit of a more structured computational model .In the factorial analysis, stay probabilities at the first stage , transition probability on the previous trial (common (70%) or rare (30%)) and outcome (loss/no loss or reward/no reward) and group (TD or BAL) were entered into three-way mixed-measures ANOVA.22 to choice behaviour, estimating free parameters for each subject separately by the method of maximum likelihood. This model contains a separate term for model-free temporal difference algorithm and model-based reinforcement-learning algorithm.In a computational-fitting analysis, we fit a previously described hybrid model 22 to choModel selection was performed with a group-level random-effect analysis of the log-evidence obtained for each tested model and subject . The estA total of 22 TD and 22 control (BAL) healthy volunteers were included in the study in a double-blind, counterbalanced design. The groups were matched by gender, age and had no differences in IQ level .F=41.595, P<0.0001; main effect of time: F=5.402, P=0.025; group × time interaction: F=41.916, P<0.0001). Post hoc analysis showed significant  reduction of serum TRP concentration in the TD group (mean±s.d.: 75.78±23.07%), but not in BAL . There was no effect of task order or an interaction of task order with TD <1.0).Post-procedure biochemical analysis showed that TD robustly decreased the TRP:ΣLNAAs ratio relative to the BAL group  and within-subject factors of task valence (reward or punishment), outcome  and transition probability on the previous trial (common (70%) or rare (30%)).F=4.22, P=0.046), outcome =17.06, P<0.0001) and transition probability =32.16, P<0.0001), but no main effect of valence =1.46, P=0.22). Across all subjects and conditions, the finding of both a main effect of outcome and outcome × transition probability interaction =28.24, P<0.0001) showed that the subjects used both model-free and model-based strategies, respectively. Importantly, the outcome × transition probability interaction (the signature of model-based learning) was significantly modulated by TD =6.21, P=0.017)), and this modulation was itself further modulated by valence =11.55, P=0.001)). There was no outcome × group interaction =0.78, P=0.38), suggesting an absence of effect of TD on model-free learning.We found main effects of group  and by group (TD versus BAL) .F=26.18, P<0.0001), transition probability =4.87, P=0.033) and an outcome × transition probability × group interaction =6.63, P=0.014) in the reward version. Post hoc separate comparisons of BAL versus TD showed a main effect of outcome =14.62, P=0.001) and an outcome × transition probability interaction =6.65, P=0.018) in the BAL group only =11.58, P=0.003) suggested a behavioural shift to the model-free strategy  =7.88, P=0.008) with a significant outcome × transition probability interaction =18.80, P<0.0001), but no main effect of outcome  =0.24, P=0.62). Overall, this result shows that subjects were aware of the task structure and demonstrated model-based behaviour in this task version. Post hoc analysis showed a mixed strategy (both model-free and model-based components in choice performance) in BAL: main effect of outcome =8.04, P=0.01) and outcome × transition probability interaction =4.77, P=0.04). For TD, there was only a significant outcome × transition interaction =12.07, P=0.002), suggesting the use of a model-based strategy in this version of the task  and task valence (reward versus punishment) had four free parameters controlling both model-based and model-free learning: learning rate α, softmax temperature β (control the choice randomness), perseverance index ρ (captures perseveration (ρ>0) or shifting (ρ<0) in the first-stage choices) and the weighting factor ω, which provides an index of the relative engagement of a model-free versus model-based behavioural choices .Similar to previous reports,ω =6.93, P=0.012) and a trend to a higher perseverance index =2.99, P=0.092). In contrast, there was no significant difference between the groups in the parameters of the loss version of the task  or between loss and reward version of the task . There were more omitted trials in the punishment version of the task in both groups, but no difference between the groups (F<1.0). Finally, there was no difference between the groups in cumulative learning in both versions of tasks <0.1, loss: F=1.31, P=0.25).The analysis of choice reaction times showed no difference between the groups on the first or second stages of the task  of time spent deliberating.30 More specifically, in the ‘average-case' reinforcement-learning model, the average reward is a signal that provides an estimation of the overall ‘goodness' or ‘badness' of the environment27 , which may help explain why the sign of the effect reverses in this condition  could also potentially affect results. Numerous studies have shown the main effect of uncertainty to be on the modulation of learning rates.37 and risk-aversion under the punishment.38 However, how the risk influences the goal-directed behaviours remains unclear, and further studies are needed to address this point.Low serotonin has been also showed to prone the risky decisions in reward condition11 by alteration of the model-based strategy. This would argue for synergy or co-operation between the DA and 5-HT systems. Nonetheless, a recent study has shown highly parallel effects of selective 5-HT depletion in rats and TD in humans on a similar task measuring increases in impulsive behaviour,39 suggesting that the effects of TD are likely to be mediated via 5-HT loss. However, our results will ultimately require confirmation using other means to reduce central 5-HT function,40 although there are no other clear-cut means to do this in human volunteers. The effects of nonspecific 5-HT receptor agents, for example, would be difficult to interpret. However, it would be of theoretical, as well as clinical, value to test the effects of enhanced 5-HT neurotransmission produced by administration of selective serotonin re-uptake inhibitors. In addition, there are no available data to clarify how DA modulates behavioural choice in the punishment condition of the task and therefore the nature of any possible interaction with the 5-HT system. However, it has been reported following either DA D2 receptor blockade or Parkinson's disease, which is characterised by diminished striatal DA neurotransmission, that there is greater attention to stimuli associated with punishment than with reward.41 We also did not show a specific effect of 5-HT depletion on model-free or habitual response in the behavioural analysis. The two-step task or model-free reinforcement learning has been suggested not to fully capture habit expression; further studies focusing on conventional over-training and testing in extinction may help clarify the effect of 5-HT depletion on habit.5Finally, in view of the proposed functional interaction of 5-HT with brain DA and evidence for the influence of DA in the balance between model-based and model-free strategies, it is possible that the effect of TD was mediated ultimately via interactions with the DA system. TD had the opposite effect to that of levodopa administration43 As model-free and model-based learning appear to have different anatomical correlates within the corticostriatal circuitry, as shown by functional neuroimaging46 and rodent lesion studies,47 it could be speculated that decreases in central 5-HT neurotransmission may affect these types of learning at different anatomical locations.The major implication of this study is that 5-HT contributes to both appetitive and aversive learning, an increasingly supported view.48 In particular, impairment of goal-directed behavioural control has been evidenced in obsessive-compulsive disorders, as well as in substance addictions and eating disorders.51Finally, our findings are also of clinical interest, as impairment of goal-directed responses has been put forward as a theoretical framework for a range of psychiatric disorders.


The purpose of this study was to prospectively evaluate the reproducibility of the proton density fat-fraction (PDFF) of the liver using the IDEAL algorithm, a quantitative confounder-corrected chemical-shift-encoded MRI method. Data were obtained from 15 volunteers on four different days. The first and the third examinations were conducted on scanner one with one-week intervals, while the second and the fourth tests were performed on scanner two with same time interval. For each test, two MR scans were performed, one before and one after a meal. Regions-of-interest measurements were manually calculated to estimate the PDFF in the right and left lobes on the PDFF images. Reproducibility was measured using the intra-class correlation coefficient (ICC). The ICCs of the PDFF in the right and left lobes were 0.935 and 0.878, respectively. The intra-scanner ICCs of the right lobe before and after a meal or at a one-week interval were 0.924 and 0.953, respectively. The inter-scanner ICCs of PDFF the next day and at a one-week interval were 0.920 and 0.864, respectively. The PDFF of liver derived from IDEAL demonstrated high intra- and inter-scanner measurement reproducibility. The PDFF of the right lobe before a meal was more reproducible than after-meal measurements. Hepatic steatosis is characterized by the excessive accumulation of lipids within hepatocytes, which is an essential feature of diffuse liver diseases, such as nonalcoholic fatty liver disease, alcoholic liver disease, viral hepatitis, genetic lipodystrophies, cystic fibrosis liver disease, and hepatotoxicity due to numerous therapeutic drugs. Liver biopsy, the current clinical gold standard for the assessment of liver fat, is invasive and produces sampling errors, which makes it a suboptimal tool to diagnose and characterize the severity of steatosis or to participate in the longitudinal follow-up of patients with liver fat.Magnetic resonance (MR) approaches can break down the liver signal into the corresponding fat and water signal components and provide more direct measurements of liver fat than those of ultrasound (US) or computed tomography (CT). Advanced MR applications measure the proton density fat-fraction (PDFF), defined as the proportion of the mobile proton density in liver tissue attributable to fat, which is a fundamental tissue property and represents the direct measurement of liver fat content. These advanced MR approaches present accurate fat quantification with a commercially available cost. A recently described complex confounder-corrected chemical shift encoded the fat-water separation method, which is based on IDEAL (Iterative Decomposition of water and fat with Echo Asymmetry and Least squares estimation), has been described for fat quantification in the liver123456in vivo, which aimed to evaluate the reproducibility of different scanners at different time scales, in different lobes of the liver, or under different levels of stomach emptiness on two respective 3.0 T MR scanners with the same algorithm, IDEAL IQ .A clinically applicable fat quantification technique must be both accurate and robust. The reproducibility of one technology refers to the proximity in agreement between a series of measurements obtained from the same subject scanned under different conditions. Therefore, the reproducibility of the fat-fraction technique is essential to ensure that the quantified data can be pooled from different time scales or from different scanners. The current prospective pilot study was conducted to assess the reproducibility of PDFF as a biomarker for liver fat content Hepatic PDFF ranged from 0.39%–9.29% (mean: 3.35% +/− 1.96%). Twelve subjects (12/15) exhibited 0%–5% PDFF, while the remaining three exhibited 6%–10% .The ICCs of PDFFs before and after meals in the right and left liver lobes across all tests were 0.935 and 0.878, respectively. The intra-scanner ICC of the right liver lobe before and after meals was 0.924. The intra-scanner ICC of the right liver lobe before meals at one-week intervals was 0.953. The inter-scanner ICCs of the PDFF performed the next day and after one week were 0.920 and 0.864, respectively .The B-A analysis’ bias (limits of agreement) of the right and left liver lobe was 0.3% (−1.1%–1.7%) and −0.1% (−1.9%–1.6%), respectively. The intra-scanner B-A of the right liver lobe before and after meals was 0.3% (−1.1%–1.8%). The intra-scanner B-A of right liver lobe at a one-week interval was −0.2% (−1.5%–1.2%). The inter-scanner B-As of the PDFF the next day and after one week were −0.1% (−1.8%–1.5%) and 0% (−2.3%–2.3%), respectively .Bland–Altman analysis revealed that the PDFF of the right liver lobe was closer than that of the left liver lobe. The B-A results and the bias (limits of agreement) of the right and left liver lobes were 0.3% (−1.1%−1.7%) and −0.1% (−1.9%−1.6%), respectively. The intra-scanner B-A of the right liver lobe before and after meals demonstrated a measurement error of (−1.1%−1.8%), which was larger than that of the right liver lobe before meals after one week (−1.5%−1.2%). Concerning the inter-scanner B-As of PDFF, the 95% limit of agreement was smaller for the PDFFs the next day (−1.8%−1.5%) than after one week (−2.3%−2.3%), which indicated that the reproducibility was slightly reduced as the interval increased .There were three major findings in the current study. First, the IDEAL IQ sequence provided PDFF measurements with high intra- and inter-scanner reproducibility in same type of 3.0-T MR scanners in healthy or mild liver steatosis subjects. Secondly, the right lobe of the liver demonstrated better reproducibility for PDFF, while the left lobe possessed acceptable reproducibility. Thirdly, food consumption might affect the reproducibility of the PDFF measurements. To the best of our knowledge, this is the first study to assess the effects of meals and the type of liver lobe on the reproducibility of MRI-based PDFF on two identical version 3.0 T MR scanners. The influence of the detected factors on intra- or inter-scanner reproducibility was significant for the validation of MRI PDFF to function as a biomarker of liver fat content.in vivo. In clinical practice, any potential error in measurement may decrease the accuracy of the diagnosis, and clinicians must assess whether the observed changes reflect the actual clinical status of a patient. To evaluate the feasibility of PDFF as a biomarker for clinical application, it is essential to investigate the reproducibility. Preliminary studies have demonstrated the accuracy of MRI-determined PDFF against spectroscopy and its within-examination repeatability8910et al.et al.’s study; the 1.5 T estimates were higher by 0.92% on average. In the current study, the bias of PDFF from two scanners on two consecutive days and at one-week intervals were −0.1% and 0%, respectively. Moreover, the imaging range in this study was different from that of Kang et al.’s study. In the current study, the PDFF maps were displayed with a range of 0–100% , while exclusion criteria included contraindications to MRI or pregnancy. All subjects provided written informed consent prior to the scan and the study was approved by the Ethics Committee of the Beijing Military General Hospital. All methods were carried out in accordance with the approved guidelines.2. Five volunteers were overweight (body mass index ≥25 kg/m2), and 10  were of normal weight.The details of each subject were recorded, including age, biological sex, body mass index (BMI), and hepatic function panel results. Eventually, fifteen healthy volunteers  were recruited, with age ranging from 22 to 57 years and a mean of 33.1 years. BMI ranged from 17.9 to 31.6 kg/mAll the participants were scanned eight times on two scanners on four respective days. The two scanners were located at two different research centres. The first and the second tests were performed on two consecutive days, whereas the third and the fourth were performed on another two continuous days. The first and third tests were conducted on scanner one, while the second and fourth tests were performed on scanner two, both with one-week intervals. Randomization was designed to perform each exam on all the subjects.Subjects underwent examinations in the supine position with a standard eight-channel torso phased-array coil centred over the liver on 3 T MR system . MR images of the liver were obtained by performing a commercial version of a chemical shift-based water-fat separation method known as IDEAL IQ, implemented with a multi-echo 3D spoiled gradient-echo acquisition to achieve six echoes with “fly-back” gradients. Both scanners were the same version of the GE Signa scanner. During each scan, water–fat separation was performed by using complex-based (T2*-IDEAL), magnitude-based, and the hybrid methods on the same source of data. PDFF images were generated from the separated water and fat images and were evaluated for each assessment.To minimize T1 effects, a low flip angle of 3° was introduced to reduce T1 bias, and a parallel imaging method, namely ARC  was performed with an acceleration factor of 2 to obtain a sufficient coverage while keeping the scan time within a single breath hold (approximately 21 s). To avoid water/fat swaps, a region-growing algorithm was employed for B0 estimation in the image reconstruction. The remaining parameters of IDEAL IQ were as follows: TR/TE1/ΔTE = 7.2/1.3/0.7 ms; receiver bandwidth = ±166.7 kHz; FOV = 26–33 cm; matrix = 160 × 160; slice thickness = 10 mm.PDFF maps were auto-generated on the host scanner after each acquisition of IDEAL IQ. All the images were independently evaluated on the AW4.5 workstation . To estimate the PDFF, one reviewer (with 8-year experience) blinded to the clinical records manually performed circular region-of-interest (ROI) measurements in the right and left liver lobes using OsiriX software on a MacOS X operating system. The PDFF images are shown in Reproducibility was measured using the intra-class correlation coefficient (ICC)The reproducibility was also assessed by calculating a Bland-Altman plot, which was generated to illustrate the agreement between the PDFF estimates derived from different times on the two scanners. The 95% limits-of agreement and the bias between two PDFF measurement values were obtained as well.How to cite this article: Wu, B. et al. Reproducibility of Intra- and Inter-scanner Measurements of Liver Fat Using Complex Confounder-corrected Chemical Shift Encoded MRI at 3.0 Tesla. Sci. Rep. 6, 19339; doi: 10.1038/srep19339 (2016).


Rapid risk stratification is a core task in emergency medicine. Identifying patients at high and low risk shortly after admission could help clinical decision-making regarding treatment, level of observation, allocation of resources and post discharge follow-up. The purpose of the present study was to determine short-, mid- and long-term mortality by plasma measurement of copeptin in unselected admitted patients.Consecutive patients >40-years-old admitted to an inner-city hospital were included. Within the first 24 hours after admission, a structured medical interview was conducted and self-reported medical history was recorded. All patients underwent a clinical examination, an echocardiographic evaluation and collection of blood for later measurement of risk markers.Plasma for copeptin measurement was available from 1,320 patients . Median follow-up time was 11.5 years (range 11.0 to 12.0 years). Copeptin was elevated .P <0.01). Three-month mortality was 14.5% (91/627) for patients with elevated copeptin compared to 3.2% (22/693) for patients with normal copeptin. Similar figures for one-year mortality and for the entire observation period were 27.6% (173/627) versus 8.7% (60/693) and 82.9% (520/527) versus 57.5% (398/693) (P <0.01 for both), respectively.Mortality within the first week was 2.7% (17/627) for patients with elevated copeptin  compared to 0.1% (1/693) for patients with normal copeptin concentrations  (Using multivariable Cox regression analyses shows that elevated copeptin was significantly and independently related to short-, mid- and long-term mortality. Adjusted hazard ratios were 2.4 for three-month mortality, 1.9 for one-year mortality and 1.4 for mortality in the entire observation period.In patients admitted to an inner-city hospital, copeptin was strongly associated with short-, mid- and long-term mortality. The results suggest that rapid copeptin measurement could be a useful tool for both disposition in an emergency department and for mid- and long-term risk assessment. An increasing number of patients are referred to emergency departments . ProlongPatients were recruited from the Copenhagen Hospital Heart Failure (CHHF) study . The priWithin the first 24 hours after admission, a structured medical interview was conducted and self-reported medical history was recorded. Additional information from previous hospital admissions was obtained from the patient files. Furthermore, all patients went through a clinical examination and an echocardiographic evaluation of the heart.After discharge, information regarding primary final diagnosis was collected from patient files. Based on this information, patients were categorized into eight separate disease categories where more rare causes for admission were categorized as ‘other’ (diagnoses of rheumatology n = 10), nephrology (n = 5), endocrinology (n = 42); and non-specific diagnoses, such as dehydration, social causes, and drug abuse) , creatinine, sodium, and potassium were obtained by standard methods. The glomerular filtration rate was estimated (eGFR) by age, creatinine, and sex using the Modification of Diet in Renal Disease (MDRD) formula .NT-proBNP measurements were taken consecutively during the last 10 months of the study using an ELISA – a two-step sandwich assay with streptavidin-coated microtiter plates  . Plasma The study complied with the Declaration of Helsinki II and was approved by the regional ethics committee of Copenhagen. Following the guidelines for provision of oral and written information, all patients gave written informed consent to participate in the study.2 –test or trend test, and Student’s t-test was used for continuous variables. Kaplan-Meier plots were used to illustrate survival curves and Cox proportional hazard model was used for initial univariate comparisons. Multivariable comparisons were performed using a Cox proportional hazard model (fitted by backward elimination using a threshold of P <0.1 for elimination) after checking assumptions of proportionality. Cox analyses were made for three-month (short-term) mortality, one-year (mid-term) mortality and mortality during the entire observation period (long-term). Due to the, by default, low mortality during the first week, Cox survival analyses were not made for one-week mortality. Areas under the curve (AUC) were calculated for copeptin in relation to one-week, three-month and one-year mortality, and mortality over the entire study period. C-statistics were calculated for all variables with independent prognostic value. Continuous variables are summarized as means and 95% confidence intervals (CI), while categorical data are summarized as frequencies and percentages. Statistical calculations were done with SPSS version 20.0 .Patients were grouped in three different ways for presentation of data and for statistical analyses: 1, elevated  versus normal copeptin concentrations; 2, quartiles of copeptin; 3, very low  versus very high (>95-percentile) copeptin concentrations. Associations between categories of variables were measured by the χBlood samples were collected from 2,294 patients. Plasma for copeptin measurement was available from 1,320 patients (36% of admitted patients) and these constitute the study population in the present paper. The remaining patients (n = 974) did not have spare plasma for copeptin analysis, but were randomly spread out across the entire study period. Patients included were 70.5 years old on average (95% CI 69.7 to 71.3 years), 59.4% were women and the mean admission time was 6.2 days (95% CI 5.6 to 6.7 days). The patients included in the present analysis were comparable to the ones without plasma samples. There was no difference between included versus excluded regarding baseline variables, comorbidity or discharge diagnoses (Table P = 0.02). Both male and female median values of copeptin were higher than previously reported values in healthy men and women [P <0.01 for both). In 47.5%  of the patients, the plasma copeptin level was higher than the previously reported 97.5 percentile in healthy subjects. The 95 percentile, 97.5 percentile and 99 percentile in this population was 89 pmol/L, 125 pmol/L and 211 pmol/L, respectively.Median plasma copeptin in the total population was 10.5 pmol/L . The median value was higher in male than in female patients , but not with sodium, P = 0.48.Baseline variables of the patients  in relation to elevated or normal plasma concentration of copeptin are presented in Table Median follow-up time was 11.5 years, (range 11.0 to 12.0 years). Complete follow-up was possible for all except one patient after one year and for all except eight patients during the entire observation period. The reason for missing information was emigration. All nine patients were censored at time of immigration. One-week mortality was 1.4% , three-month mortality was 8.6% , one-year mortality was 17.7% , and 69.3%  died during the entire study period.a), for quartiles of copeptin (b), and for patients with very low (<5-percentile) and very high copeptin (>95-percentile) (c). Mortality within the first week, three-month mortality, one-year mortality, and mortality for the entire observation period for different groupings of copeptin appear in Table Figure Using multivariable Cox regression analyses, we examined the prognostic value of copeptin in relation to elevated values of copeptin. All 20 variables from Table th-percentile were significantly and independently related to short-, mid- and long-term mortality in the entire population when the above-mentioned variables were included in the model.HRs for patients with elevated copeptin in the different disease categories are shown in Figure Table P = 0.04) and for three-month mortality (0.72 (95% C.I. 0.70 to 0.75) versus 0.77 (95% CI 0.73 to 0.81) versus 0.79 (95% CI 0.75 to 0.83), P = 0.01), respectively. There was no significant effect on AUCs of adding copeptin to the fully adjusted model for one-year mortality or for the entire observation period.Receiver operating characteristic (ROC) analyses show that the AUC for copeptin to predict one-week, short-, mid- and long-term mortality was 0.84 (95% CI 0.77 to 0.92), 0.75 (95% CI 0.70 to 0.80), 0.71 (95% CI 0.67 to 0.74) and 0.70 (0.67 to 0.73), respectively. Comparing a model with age, gender and comorbidity (model 1) to a model containing all variables included in the final Cox model , gender, age, liver disease, potassium and hemoglobin) (model 2) and to the fully adjusted model with copeptin added (model 3), we found a significant increase in AUCs for one-week mortality (0.70 (95% C.I. 0.65 to 0.76) versus 0.80 (95% CI 0.73 to 0.88) versus 0.85 (95% CI 0.77 to 0.93), To the best of our knowledge, this study is the first to determine the prognostic value of copeptin measurement in unselected patients ≥40- years old admitted to both medical and surgical departments. Copeptin measurement had a strong and independent prognostic importance for all patients and seems not to be related to any specific diagnosis. Patients with low copeptin concentrations had an excellent short-term prognosis with no fatal outcomes within the first week and only very few deaths within three months. On the other hand, high copeptin concentrations identified a subgroup of patients with a very high risk of short-term mortality.et al. published a slightly higher 97.5 percentile  based on data from the Gutenberg health study [The cut-off level for elevated copeptin in the present analysis is based on the 97.5 percentile from the original assay article(4). Recently Keller th study . This poOur findings corroborate and extend previous studies investigating the prognostic role of copeptin in different settings. In patients with ischemic heart disease, copeptin has been shown to have a high prognostic value with adjusted HRs comparable to the results from the present study ,12. SimiThe strengths of this study are the size of the cohort, its prospective and relative unselected nature, and the long follow-up period. A cohort of unselected patients admitted to a hospital is unique and makes it possible to test the role of copeptin under various medical and surgical conditions. The heterogeneity of the different disease subgroups and limited number of patients within each group does not allow firm conclusions but serves to generate further hypotheses. Only all-cause mortality was registered. Causes of death and admissions would have been other interesting endpoints, but their validity is suboptimal in the registries. The study was a single center study and this could potentially cause problems with generalizability. This concern is, however, partly countered by the fact that the hospital is placed in a part of Copenhagen that includes both high income and low income areas as well as inner city and surburban /country areas. Blood samples were stored for 12 years at –80°C, which could introduce a problem with degradation. However, stability analyses of copeptin have shown that copeptin is a very stable peptide, and no degradation is seen after several cycles of freeze/thaw or after as much as seven days at room temperature ,33. EvenBlood was available from only 53% of the originally included patients, the main reason being a lack of spare plasma after blood had been used for other purposes. However, we did not identify any systematic reason for this selection, and there were no differences in baseline variables or mortality between included and not included patients. Some selection bias in the original cohort was inevitable due to missing informed consent from confused, mentally disabled and frail patients. The mean age of the population in the study is high (74 years) and there was a high prevalence of comorbidity. Therefore, the results of this study cannot without caution be extrapolated to younger or non-hospitalized populations.The results of this study suggest that copeptin could be a very useful tool for disposition in an emergency department. Knowing that patients had a seven-day mortality of more than 10% or a one-year mortality of less than 3% might influence the clinician’s decisions regarding observation and treatment in the emergency department. Studies evaluating an intervention  following measurement of copeptin could show if the findings in the present study could be transformed to a changed and improved clinical practice.In patients ≥40 years of age admitted to an inner-city hospital, copeptin in plasma was strongly associated with short-, mid- and long-term mortality. Having very high or very low copeptin identifies a subgroup of patients, respectively, with very poor and very good short-term prognosis. Copeptin seems to be valuable in the most frequent disease entities.AUC: area under the curve; AVP: arginine vasopressin; CHHF: Copenhagen Hospital Heart Failure; CI: confidence interval; CRP: C-reactive protein; eGFR: estimated glomerular filtration rate; ELISA: enzyme-linked immunosorbent assay; HR: hazard ratio; NT-proBNP: N-terminal pro-brain natriuretic peptide.The authors declare that they have no competing interests.MB and VK collected data and critically revised the manuscript. JPG, SB, MD, OWN and HN participated in drafting of the manuscript and interpretation of the data and critically revised the manuscript. LK participated in drafting of the manuscript, data analyses and interpretation of the data and critically revised the manuscript. KI wrote the first draft of the manuscript, performed the analyses and interpretation of the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/12/80/prepub